The demonstration that mature adult cells can be reprogrammed to create induced pluripotent stem cells (iPSCs), which are like embryonic stem cells with respect to their self-renewal and differentiation capacities, has sparked a great deal of interest in this technology. The initial methods for reprogramming focused on delivery of four genes (Oct4, Sox2, and either Klf4 plus c-Myc, or NANOG plus Lin 28) using genetic transduction methods that potentially can create mutations and/or tumors. More recently, it was demonstrated that delivery of 4 proteins to adult cells could also generate iPSCs. However, reprogramming directly with proteins suffers from two major problems: very low efficiency of iPSC generation (0.001-0,006%) and extremely high cost of reprogramming proteins, making it not practical for use on a routine basis. The overall objective of the current proposal is to apply 2 novel technologies to make generation of iPSCs more practical, simple and affordable. The first is the QQ-protein transduction technology, which is a simple and highly efficient method to specifically deliver purified reprogramming proteins into the nuclei of cells for initiating and maintaining constant protein reprogramming for iPSC generation. The second is a bacterial expression system capable of producing gram quantities of protein combined with is a novel in vivo protein refolding technique to produce large quantity of purified, functional reprogramming proteins at a very low cost. The application of these new technologies is expected to significantly enhance the efficiency of generating iPSC by 1000-fold. In addition, it will make creation of iPSCs much simpler and will significantly reduce the cost so the technology can be used by many more investigators than current technologies. This will have a revolutionary effect on iPSC research and on regenerative medicine. The commercial products and services upon which the field depends will have a rapidly growing market. The commercial benefits of this project will allow TechTown Ventures to contribute to the economic recovery of Southeast Michigan. PUBLIC HEALTH RELEVANCE: The results of this research will make creation of induced pluripotential stem cells (iPSC) without use of genetic material simpler, safer and affordable. These advances will put the technology in reach of most researchers and thereby stimulate the creation of new cell lines for studies on many different diseases. They will also make it possible to create pluripotential stem cells from a patient's normal cells that can be used clinically to replace the diseased or injured cells.